Recent development of the polymerase chain reaction ("PCR") has provided an important tool for the detection of nucleic acid sequences present at low concentrations (Mullis, K. B. et al., U.S. Pat. Nos. 4,683,195 and 4,683,202). In PCR, a target sequence having boundaries defined by two oligonucleotide extension primers, is amplified through repeated enzymatic cycles to provide additional templates for further amplification reactions. Accordingly, a small number of target sequences can be exponentially amplified and readily detected.
A major limitation of PCR lies in the extensive generation of by-products produced as a result of non-specific priming events, e.g., random priming of the nucleic acid template and/or self priming of the extension primers. Thus, when a high number of amplification cycles are required to amplify a target sequence present at a relatively low concentration, the products of non-specific priming events significantly impede PCR sensitivity.
An additional, related limitation of PCR is the requirement for a separation step prior to detection of the amplified target. According to standard PCR conditions, separation of the amplified target sequence from the products of non-specific priming events is a prerequisite for detection of the amplified target sequence. The absence of a homogenous amplification reaction, i.e., an reaction in which amplification and detection take place in the same reaction vessel has been an obstacle in automating the PCR procedure. In addition, the requirement for a separation step also subjects the PCR mixture to potential contamination resulting from the separation procedure. The likelihood of contamination severely limits the potential application of PCR in routine clinical diagnosis.
Attempts have been reported to develop a homogeneous assay for amplification and detection. One such attempt is described by Holland et al. (1991, Proc. Natl. Acad. Sci. USA 88:7276, hereby incorporated by reference) in an assay utilizing the 5'.fwdarw.3' exonuclease activity of Taq polymerase to generate a detection signal concomitantly with PCR amplification. However, a subsequent separation step is required to detect the exonuclease generated signal. Higuchi et al. (1992, Bio/Technology 10: 413, hereby incorporated by reference) describes a homogeneous detection process based on monitoring enhanced fluorescence of ethidium bromide when it is intercalated into double stranded amplification product. Such a detection process is not target sequence specific, thus vulnerable to interferences from the presence of any double stranded DNA. A different approach is to use fluorescence polarization to detect hybridization of a fluorescence nucleic acid probe or fluorescence primer extension products (Garmen, A. J. et al., European Patent Office Publication No. 382,433). This detection process is able to distinguish the hybridized or extended probe, which has a higher molecular weight (thus decreased fluorescence polarization), from the unreacted probe. However, the presence of the unhybridized probe with higher polarization strongly influences the observed polarization, thus giving a high background.
Non-radiative energy transfer by close proximity of two fluorescent moieties can be used as an effective signal detection mode. Homogeneous immunoassays based on fluorescence energy transfer have been described (Patel. et. al., European Patent Application No. 137,515). Fluorescence energy transfer has also been designed for detecting nucleic acid hybridization (Heller et al., European Patent Application No. 070,685 and U.S. Pat. No. 4,996,143). In such applications, a fluorescence energy transfer signal is generated when probes carrying different fluorescent moieties are brought to the close physical vicinity as a result of hybridization to a strand of the target nucleic acid.